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Biomedical subjects

V H Engelhard

Publications and source records attributed to V H Engelhard.

At least 73 records · Page 4Linked to original sources
PubMed1994

Inhibition of T cell activation by a monoclonal antibody reactive against the alpha 3 domain of human MHC class I molecules.

A long term goal of investigators working in our laboratory has been to develop new mAbs for use as immunosuppressive agents. As a means toward achieving this goal, several new mAbs (hybridomas) have been developed by screening fusions for supernatants that possess T cell-inhibitory properties. Of these new mAbs, one mAb, designated 5H7, has been shown to possess both a unique combination of specificity for a monomorphic determinant of the alpha 3 domain of human class I MHC heavy chains and highly potent T cell inhibitory properties. mAb 5H7 profoundly inhibited T cell proliferation in response to anti-CD3 mAb in primary or secondary allogenic MLR or in primary human anti-mouse xenogenic MLR. mAb 5H7 inhibited expression of an early T cell activation marker, Leu23 (CD69); expression of IL-2Rs; and IL-2 production by both CD4+ and CD8+ T cells. mAb 5H7 inhibited IL-2 release by the Jurkat (E6-1) human T cell leukemia line in response to immobilized anti-CD3 mAb, thus, providing further evidence that 5H7 can inhibit activation directly at the level of the T cell. 5H7 profoundly blocked CD3-dependent (anti-CD3, anti-CD3 plus PMA, or anti-CD3 plus anti-CD28) pathways, but only partially blocked a CD3-independent (anti-CD28 plus PMA) pathway of T cell activation. In conclusion, class I MHC molecules that are expressed on the T cell may regulate early TCR/CD3-dependent signaling events. In addition, 5H7 mAb may provide a reagent for suppression of cellular immunity in vivo.

Antibodies, Monoclonal↗
PubMed1994

Cytotoxic T-lymphocyte response to autologous human squamous cell cancer of the lung: epitope reconstitution with peptides extracted from HLA-Aw68.

Cytotoxic T-lymphocytes (CTLs) specific for autologous human squamous cell cancer of the lung were generated by stimulation of peripheral blood lymphocytes with autologous tumor cells in vitro. The CTL line was >97% CD3+, CD8+, CD16- and produced tumor necrosis factor-alpha, gamma-interferon, and granulocyte-macrophage colony-stimulating factor after stimulation with autologous tumor. The CTLs lysed autologous tumor but failed to recognize autologous or histocompatibility leukocyte antigen-matched lymphoid cells, K562, or allogeneic tumor cells of several histological types. Antibody-blocking studies suggested that the CTLs recognized one or more antigens presented by the class I major histocompatibility complex molecule Aw68. To characterize these antigens further, histocompatibility leukocyte antigen Aw68 molecules were extracted from the squamous cell cancer of the lung tumor line by immunoaffinity chromatography, and the associated peptides were eluted in acid and separated by reversed-phase high-performance liquid chromatography. Reconstitution of the CTL epitope was evaluated by adding these peptides to autologous Epstein-Barr virus-transformed B-cells. Two peaks of reconstituting activity were observed, suggesting that these CTLs recognize at least two Aw68-associated peptides. This study confirms the existence of a CTL response against autologous human squamous cell cancer of the lung and suggests that this CTL response is directed against peptide epitopes presented by the class I major histocompatibility complex molecules. It is anticipated that this approach will permit identification of peptide epitopes for lung cancer-specific CTLs.

Carcinoma, Squamous Cell↗
PubMed1994

Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines.

Of several thousand peptides presented by the major histocompatibility molecule HLA-A2.1, at least nine are recognized by melanoma-specific cytotoxic T lymphocytes (CTLs). Tandem mass spectrometry was used to identify and to sequence one of these peptide epitopes. Melanoma-specific CTLs had an exceptionally high affinity for this nine-residue peptide, which reconstituted an epitope for CTL lines from each of five different melanoma patients tested. Recognition by multiple CTL lines suggests that this may be a promising candidate for use in peptide-based melanoma vaccines.

Amino Acid Sequence↗
PubMed1994

Association of human class I MHC alleles with the adenovirus E3/19K protein.

A panel of HLA-A and -B locus products was analyzed for their ability to associate with the adenovirus E3/19K (E19) protein in a co-immunoprecipitation assay. Three general categories of binding were identified. HLA-A2.1 and -B7 bind very well to E19. Compared with A2.1, 6- to 30-fold less E19 was associated with HLA-A3, -A1, and -Aw69; 50- to 150-fold less E19 was associated with HLA-Aw68, -B27, and -Bw58. Digestion with endoglycosidase H indicated that all levels of association resulted in inhibition of intracellular transport and processing, however, a fraction of Aw68, B27, and Bw58 escaped from intracellular retention. In contrast to the human class I molecules analyzed, transport of the murine H-2Dd molecule was not inhibited in the presence of E19. Hybrid class I molecules, in which exons encoding domains of A2.1 and H-2Dd had been exchanged, were used to define the regions of A2.1 required for E19 association. The alpha 1 and alpha 2 domains of A2.1 contain the minimum residues necessary for both stable association with E19 and subsequent inhibition of transport. A hybrid construct containing only the alpha 2 domain of A2.1 associated weakly with E19, but its post-translational processing was completely inhibited. In contrast, although a construct containing only the alpha 1 domain of A2.1 also associated weakly with E19, its intracellular transport was slowed rather than completely inhibited. Taken together, these results indicate that residues in both the alpha 1 and alpha 2 domains of A2.1 and Dd can influence stable binding of E19, with the phenotypic changes dominated by the origin of the alpha 2 domain.

Adenovirus Early Proteins↗
PubMed1994

Naturally processed peptides longer than nine amino acid residues bind to the class I MHC molecule HLA-A2.1 with high affinity and in different conformations.

An equilibrium binding assay was used to directly measure the relative affinities of naturally processed 9-mer, 10-mer, and 12-mer peptides for the human class I MHC molecule HLA-A2.1. The peptides exhibited a range of affinities with IC50 values of 11 to 214 nM. The mode of interaction between these peptides and HLA-A2.1 was examined using peptides in which Asp had been substituted for suspected anchor residues. Regardless of length, the previously identified Leu at position 2 relative to the amino terminus was critical for peptide binding. While the carboxyl terminal residue was also critical for the binding of a 9-mer peptide, it was much less important in the binding of longer peptides. Additional residues close to the carboxyl terminus that contained aliphatic hydrocarbon side chains were of similar or greater importance in peptide binding. In addition, residue at position 3 also appeared to be important for the binding of longer peptides. The data suggest that different naturally occurring longer peptides can bind in different conformations to class I MHC molecules. While one of these is similar to the kinked conformation described by others, another conformation would involve an extension of the carboxyl terminus out of the class I binding site. The ability of MHC molecules to accommodate the same peptide in different conformations would appear to have distinct advantages to the immune system.

Amino Acid Sequence↗
PubMed1994

Structure of peptides associated with MHC class I molecules.

Recent progress in understanding the structures of MHC class I molecules and the peptides that they bind has led to a generalized model for peptide binding, and an understanding of allelic specificity. Prediction on the basis of motifs and new techniques for peptide analysis have recently resulted in the identification of several peptides that comprise epitopes for antigen-specific T cells.

Amino Acid Sequence↗
PubMed1994

Direct analysis of tumor-associated peptide antigens.

Adoptive immunotherapy with tumor-specific cytotoxic T lymphocytes (CTLs) can induce tumor regressions in animals and in human cancer patients. Antigens recognized by CTLs from cancer patients are being sought as possible immunogens, a number of which have been identified during the past year. The ultimate result may be the development of novel peptide-based immunotherapies and a new understanding of the T-cell response to human cancer.

Amino Acid Sequence↗
PubMed1994

Structure of peptides associated with class I and class II MHC molecules.

Class I and class II molecules encoded by genes within the major histocompatibility complex play a central role in regulation of immune responses through their ability to bind and display small peptides derived from foreign antigens. Within the last few years, considerable progress has been made in understanding the structures of class I and class II MHC molecules, as well as the features of the peptides that are their principal ligands. This review summarizes this information and describes how it accounts for both the specificity and degeneracy of peptide binding. It also considers how the origin and structural features of peptides that have been isolated from MHC molecules, so-called "naturally processed" peptides, have provided insight into the pathways through which the peptides are produced. Finally, the use of new structural information and techniques for peptide characterization for the identification of peptides that comprise epitopes for individual antigen-specific T cells are considered.

Amino Acid Sequence↗
PubMed1993

Direct identification of an endogenous peptide recognized by multiple HLA-A2.1-specific cytotoxic T cells.

An endogenous peptide recognized by a murine T-cell clone specific for the human class I major histocompatibility complex-encoded molecule HLA-A2.1 was identified through the use of microcapillary high-performance liquid chromatography coupled with electrospray-ionization tandem mass spectrometry. The peptide was associated with HLA-A2.1 on both normal cells and the antigen-processing-mutant cell line T2. This observation demonstrates that a processing mechanism other than that involving the transporter associated with antigen processing (TAP) proteins 1 and 2 can produce peptides that can be recognized by T cells. The peptide was also recognized by four other independently derived murine HLA-A2.1-specific murine T-cell clones. This suggests that xenogeneic responses are directed at a restricted subset of major histocompatibility complex product-associated peptides. Finally, quantitation of this peptide in cell extracts using mass spectrometry showed it to be among the most dominant HLA-A2.1 associated species on human lymphoid cells. The potential relevance of this observation to models of alloreactivity will be discussed. The methodology described should be generally useful for the identification of peptide epitopes recognized by alloreactive, tumor-specific, and autoimmune T cells.

Amino Acid Sequence↗
PubMed1993

Characteristics of endogenous peptides eluted from the class I MHC molecule HLA-B7 determined by mass spectrometry and computer modeling.

Microcapillary HPLC electrospray ionization tandem mass spectrometry was used to sequence 15 peptides eluted from HLA-B7. Sequence alignment implicated four peptide positions in specific interactions with the class I molecule, and their importance was confirmed using synthetic peptides. Because no crystal structure for HLA-B7 was available, computer-assisted modeling was used to understand novel aspects of peptide binding specificity and to accurately predict the effect of defined changes in peptide structure. The results demonstrate that mass-spectrometric sequencing coupled with computer-assisted modeling can be used in the absence of a crystal structure to make accurate predictions concerning requirements for peptide binding to class I molecules. These techniques may be valuable to predict or engineer T cell epitopes.

Amino Acid Sequence↗
PubMed1993

Recognition of human melanoma cells by HLA-A2.1-restricted cytotoxic T lymphocytes is mediated by at least six shared peptide epitopes.

HLA-A2.1-associated peptides were extracted from human melanoma cell lines and used to study epitopes for melanoma-specific HLA-A2.1-restricted CTL. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic A2.1+ melanoma cells. These CTL lysed autologous melanoma plus four allogeneic HLA-A2.1+ melanomas, including an HLA-A2.1-transfected melanoma. K562, HLA-A2- melanomas and HLA-A2+ nonmelanomas were not lysed. HLA-A2.1 molecules were purified from human melanoma cell lines by immunoaffinity column chromatography of detergent-solubilized cell pellets. Peptides bound to the MHC molecules were acid eluted and fractionated by reversed phase HPLC. Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by addition to the HLA-A2.1+ Ag-processing mutant, 721.174XCEM.T2 (T2). Five peaks of reconstitution were observed. Second dimension HPLC separations of reconstituting fractions revealed evidence for two distinct reconstituting peptides within one of these peaks. Based on these data, a minimum of six distinct peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL are present on these different melanoma lines. These data document the presence of multiple peptide-defined CTL epitopes that are shared by at least three unrelated human melanoma cell lines.

Cell Line↗
PubMed1992

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.

Animals↗
PubMed1992

Species specificity and augmentation of responses to class II major histocompatibility complex molecules in human CD4 transgenic mice.

Murine T cell responses to human class II major histocompatibility complex (MHC) molecules were shown to be a minimum of 20-70-fold lower than responses to allogeneic molecules. Transgenic mice expressing slightly below normal (75-95%) or very high (250-380%) cell surface levels of human CD4 were utilized to determine whether this was due to a species-specific interaction between murine CD4 and class II molecules. Human CD4 was shown to function in signal transduction events in murine T cells based on the ability of anti-human CD4 antibody to synergize with suboptimal doses of anti-murine CD3 antibody in stimulating T cell proliferation. In mice expressing lower levels of human CD4, T cell responses to human class II molecules were enhanced up to threefold, whereas allogeneic responses were unaltered. In mice expressing high levels of human CD4, responses to human class II molecules were enhanced at least 10-fold, whereas allogeneic responses were between one and three times the level of normal responses. The relatively greater enhancement of the response to human class II molecules in both lines argues for a preferential interaction between human CD4 and human class II molecules. In mice expressing lower levels of human CD4, responses to human class II molecules were blocked by antibodies to CD4 of either species, indicating participation by both molecules. In mice expressing high levels of human CD4, responses to both human and murine class II molecules were almost completely blocked with anti-human CD4 antibody, whereas anti-murine CD4 antibody had no effect. However, anti-murine CD4 continued to synergize with anti-CD3 in stimulating T cell proliferation in these mice. Thus, overexpression of human CD4 selectively impaired the ability of murine CD4 to assist in the process of antigen recognition. The ability of human CD4 to support a strong allogeneic response under these conditions indicates that this molecule can interact with murine class II molecules to a significant extent. Despite the fact that human CD4 appeared to be the only functional coreceptor in these mice, responses to human class II molecules were still much lower than those to murine class II alloantigens. This indicates that species-specific interactions between class II molecules and CD4 expressed on peripheral T cells are not sufficient to account for the low xenogeneic response and that intrinsic differences in T cell receptor structures or the need for species specificity in the interaction between CD4 and class II molecules during positive selection are also important.

Animals↗
PubMed1992

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.

Animals↗
PubMed1992

Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.

Antigens recognized by T cells are expressed as peptides bound to major histocompatibility complex (MHC) molecules. Microcapillary high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to fractionate and sequence subpicomolar amounts of peptides isolated from the MHC molecule HLA-A2.1. Of 200 different species quantitated, eight were sequenced and four were found in cellular proteins. All were nine residues long and shared a distinct structural motif. The sensitivity and speed of this approach should enhance the analysis of peptides from small quantities of virally infected and transformed cells as well as those associated with autoimmune disease states.

Amino Acid Sequence↗
PubMed1992

HLA-A2.1-associated peptides from a mutant cell line: a second pathway of antigen presentation.

Peptides extracted from HLA-A2.1 class I major histocompatibility complex (MHC) molecules expressed on the antigen processing mutant CEMx721.174.T2 were characterized by electrospray ionization-tandem mass spectrometry. Only seven dominant peptides were found, in contrast to over 200 associated with HLA-A2.1 on normal cells. These peptides were derived from the signal peptide domains of normal cellular proteins, were usually larger than nine residues, and were also associated with HLA-A2.1 in normal cells. These results suggest that proteolysis of signal peptide domains in the endoplasmic reticulum is a second mechanism for processing and presentation of peptides for association with class I molecules.

Amino Acid Sequence↗